Insight into the mechanism behind the activation phenomenon of lipase from Thermus thermophilus HB8 in polar organic solvents

Q2 Chemical Engineering

Journal of Molecular Catalysis B-enzymatic Pub Date : 2016-11-01 DOI:10.1016/j.molcatb.2017.03.002

Hao Cao , Kaili Nie , Haijun Xu , Xin Xiong , Rumen Krastev , Fang Wang , Tianwei Tan , Luo Liu

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引用次数: 10

Abstract

A thermophilic lipase from Thermus thermophilus (lipaseTt) was heterologously overexpressed in Escherichia coli BL21(DE3). The tolerance of (lipaseTt) towards polar organic solvents and the associated activation phenomena were investigated. The catalytic efficiency (Kcat/K_M) of lipaseTt in 56% DMSO was up to 25.64 times higher than that in phosphate buffer. Molecular dynamic (MD) simulations were carried out to investigate the activation mechanism of the lipaseTt by polar organic solvents. After refining the homology modeling of lipaseTt by using MD-simulation, a new secondary structure in the lid region indicates a rigid structure, corresponding to its nature of thermostability. Subsequently, the loss of a secondary structure of lipaseTt in the presence of polar organic solvent was observed in both MD-simulation and Circular dichroism spectra. The results of MD-simulation demonstrate that the overall structure maintained stable with or without polar organic solvent; however, the lid region was found more flexible in the presence of polar organic solvent. The flexible lid facilitates the substrate to access the catalytic site inside the lipase and the lipase displays enhanced activity in the presence of a polar organic solvent.

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研究嗜热热菌HB8脂肪酶在极性有机溶剂中的活化机制

一种来自嗜热热菌的嗜热脂肪酶(lipaseTt)在大肠杆菌BL21(DE3)中异源过表达。研究了(lipaseTt)对极性有机溶剂的耐受性及其活化现象。lipaseTt在56% DMSO中的催化效率(Kcat/KM)比在磷酸盐缓冲液中的催化效率高25.64倍。通过分子动力学(MD)模拟研究极性有机溶剂对脂质醚的活化机理。利用MD-simulation对lipaseTt的同源性建模进行完善后，在盖子区域发现了一个新的二级结构，表明其为刚性结构，与其热稳定性的性质相对应。随后，在md模拟和圆二色光谱中观察到lippasett在极性有机溶剂存在下的二级结构损失。md模拟结果表明，无论是否加入极性有机溶剂，整体结构都保持稳定;然而，在极性有机溶剂的存在下，发现盖子区域更具柔韧性。所述柔性盖便于所述底物进入所述脂肪酶内部的催化位点，所述脂肪酶在极性有机溶剂存在下显示增强的活性。

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来源期刊

Journal of Molecular Catalysis B-enzymatic 生物-生化与分子生物学

CiteScore

2.58

自引率

0.00%

发文量

审稿时长

3.4 months

期刊介绍： Journal of Molecular Catalysis B: Enzymatic is an international forum for researchers and product developers in the applications of whole-cell and cell-free enzymes as catalysts in organic synthesis. Emphasis is on mechanistic and synthetic aspects of the biocatalytic transformation. Papers should report novel and significant advances in one or more of the following topics; Applied and fundamental studies of enzymes used for biocatalysis; Industrial applications of enzymatic processes, e.g. in fine chemical synthesis; Chemo-, regio- and enantioselective transformations; Screening for biocatalysts; Integration of biocatalytic and chemical steps in organic syntheses; Novel biocatalysts, e.g. enzymes from extremophiles and catalytic antibodies; Enzyme immobilization and stabilization, particularly in non-conventional media; Bioprocess engineering aspects, e.g. membrane bioreactors; Improvement of catalytic performance of enzymes, e.g. by protein engineering or chemical modification; Structural studies, including computer simulation, relating to substrate specificity and reaction selectivity; Biomimetic studies related to enzymatic transformations.